Inhibition of tumor growth and metastasis by photoimmunotherapy targeting tumor-associated macrophage in a sorafenib-resistant tumor model

Accepted Manuscript Inhibition of tumor growth and metastasis by photoimmunotherapy targeting tumorassociated macrophage in a sorafenib-resistant tumor model Chenran Zhang, Liquan Gao, Yuehong Cai, Hao Liu, Duo Gao, Jianhao Lai, Bing Jia, Fan Wang, Zhaofei Liu, Ph.D. PII:

S0142-9612(16)00035-1

DOI:

10.1016/j.biomaterials.2016.01.027

Reference:

JBMT 17303

To appear in:

Biomaterials

Received Date: 28 November 2015 Revised Date:

11 January 2016

Accepted Date: 12 January 2016

Please cite this article as: Zhang C, Gao L, Cai Y, Liu H, Gao D, Lai J, Jia B, Wang F, Liu Z, Inhibition of tumor growth and metastasis by photoimmunotherapy targeting tumor-associated macrophage in a sorafenib-resistant tumor model, Biomaterials (2016), doi: 10.1016/j.biomaterials.2016.01.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Inhibition of tumor growth and metastasis by photoimmunotherapy targeting tumor-associated macrophage in a sorafenib-resistant

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tumor model Chenran Zhang1, Liquan Gao1, Yuehong Cai2, Hao Liu1, Duo Gao1, Jianhao Lai1, Bing Jia1, Fan Wang1,3, Zhaofei Liu1*

Medical Isotopes Research Center and Department of Radiation Medicine, School of

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Basic Medical Sciences, Peking University Health Science Center, Beijing 100191,

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China 2

Division of Medical Sciences, Weifang People’s Hospital, Weifang 261000, China

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Interdisciplinary Laboratory, Institute of Biophysics, Chinese Academy of Sciences,

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Beijing 100101, China

*Corresponding author: Zhaofei Liu, Ph.D., Medical Isotopes Research Center and

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Department of Radiation Medicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China. Tel./fax: +86-10-82802871; E-mail: [email protected]

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Graphical Abstract

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Abstract Tumor-associated macrophages (TAMs) play essential roles in tumor invasion and

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metastasis, and contribute to drug resistance. Clinical evidence suggests that TAM levels are correlated with local tumor relapse, distant metastasis, and poor prognosis in patients. In this study, we synthesized a TAM-targeted probe (IRD-αCD206) by

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conjugating a monoclonal anti-CD206 antibody with a near-infrared phthalocyanine

dye. We then investigated the potential application of the IRD-αCD206 probe to

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near-infrared fluorescence (NIRF) imaging and photoimmunotherapy (PIT) of tumors resistant to treatment with the kinase inhibitor sorafenib. Sorafenib treatment had no effect on tumor growth in a 4T1 mouse model of breast cancer, but induced M2 macrophage polarization in tumors. M2 macrophage recruitment by sorafenib-treated

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4T1 tumors was noninvasively visualized by in vivo NIRF imaging of IRD-αCD206. Small-animal single-photon emission computed tomography (SPECT)/CT and intratumoral microdistribution analysis indicated TAM-specific localization of the

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IRD-αCD206 probe in 4T1 tumors after several rounds of sorafenib treatment. Upon light irradiation, IRD-αCD206 suppressed the growth of sorafenib-resistant tumors. In

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vivo CT imaging and ex vivo histological analysis confirmed the inhibition of lung metastasis in mice by IRD-αCD206 PIT. These results demonstrate the utility of the

IRD-αCD206 probe for TAM-targeted diagnostic imaging and treatment of tumors that are resistant to conventional therapeutics. Keywords: Tumor-associated macrophage (TAM); Tumor microenvironment; Molecular imaging; Photoimmunotherapy; Tumor metastasis 3

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1. Introduction Traditional cancer treatments such as radio- and chemotherapy are associated with

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serious side effects and drug resistance [1]. Although there are many drugs that maximize treatment efficacy while minimizing toxicity, local tumor recurrences and

distant metastasis are common, and may eventually lead to drug resistance and

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treatment failure [2, 3].

Sorafenib (BAY 43-9006, Nexavar) is a small molecule tyrosine kinase inhibitor

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that is approved for the treatment of metastatic renal cell carcinoma and advanced hepatocellular carcinoma [4]. It has also been investigated for the treatment of lung, breast, and other cancers [5, 6]. Several clinical studies have found that the patient response rate to this drug is quite low, with cancers becoming sorafenib-resistant after

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several rounds of therapy [7, 8]. Therefore, novel treatments for tumors, especially those that are drug-resistant, are urgently needed. During cancer development and treatment, tumors are infiltrated by myeloid cells,

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such as macrophages (tumor-associated macrophages; TAMs), which are recruited in their monocyte precursor form from the peripheral blood [9]. TAM stimulates tumor

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progression by facilitating angiogenesis, invasion, and metastasis, and suppressing antitumor immunity [10, 11]. TAM levels are correlated with poor prognosis in various human cancers and are a major factor in the development of resistance to chemo- or radiotherapy, as well as in tumor recurrence [12, 13]. As such, characterization of macrophage infiltration in tumors may be used to predict patient outcome and evaluate responses to therapy [14]. 4

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TAMs are typically designated as “alternatively activated” non-inflammatory M2-type macrophages, in contrast to the “classically activated” inflammatory “M1”

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type [15]. TAMs can serve as therapeutic targets or markers for imaging in the evaluation of tumor progression [16]. M2 macrophages specifically express CD206, also known as macrophage mannose receptor [17-19]. CD206-targeted single-photon

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emission computer tomography (SPECT) or optical imaging can potentially be used

as a means of noninvasively visualizing the TAM infiltration into tumors in vivo [20,

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21].

Antibody- and phthalocyanine dye-based photodynamic therapy (PDT), also known as photoimmunotherapy (PIT) [22], uses the near-infrared phthalocyanine dye IRDye700 as a photosensitizer in conjunction with an antibody that serves as a

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delivery vehicle for tumor-specific near-infrared fluorescence (NIRF) imaging. Upon light irradiation, PIT selectively destroys malignant cells targeted by antibodies while sparing normal adjacent tissue. The efficacy of PIT has been demonstrated in several

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tumor models [23-26].

In this study, we synthesized a CD206-targeted PIT probe by conjugating a

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monoclonal anti-CD206 antibody with IRDye700 and evaluated its effectiveness in vitro and in vivo. We investigated whether the PIT probe could be used to noninvasively visualize and image TAM recruitment to tumors as well as treat tumors that are resistant to sorafenib therapy. Our findings provide information that can be useful for predicting therapeutic response and evaluating drug resistance.

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2. Materials and methods 2.1. Cell culture and animal model

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The 4T1 murine breast cancer and RAW264.7 murine macrophage cell lines were purchased from American Type Culture Collection. Cells were cultured in

RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum at 37°C under

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a humidified atmosphere containing 5% CO2.

All animal procedures were performed in accordance with the Guidelines of

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Peking University Animal Care and Use Committee. The 4T1 tumor-bearing mouse model was established by subcutaneous injection of 1×106 cells into the right hind legs of female BALB/c mice (5 weeks of age; Department of Laboratory Animal Science, Peking University). The tumor growth was measured using a caliper, and the

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tumor volume was calculated using the formula: volume = length× width2/2.

2.2. Sorafenib treatment

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The 4T1 tumor-bearing mice with a tumor size of 100–150 mm3 were selected for in vivo sorafenib treatment studies. A sorafenib dose of 20–50 mg/kg/day has been

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used in other studies [27-29]. Therefore, 4T1 mice were segregated into four groups (n = 18 per group), and were administered with 0 (vehicle control), 20, 40, or 60 mg sorafenib/kg/day (in 1:1 propylene glycol/water) for 6 days (days 0–5) by gavage. Tumor size and body weight were measured every other day. On day 6, three mice from each group were sacrificed, and their tumors were harvested. After cutting on a freezing microtome, tumor tissues were stained with an anti-CD206 antibody (clone 6

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C068C2, IgG2a; BioLegend, San Diego, CA) for 1 h at room temperature and then visualized with dye-conjugated secondary antibody under a Leica TCS-NT confocal

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microscope (Wetzler, Heidelberg, Germany). Meanwhile, a total of 10 mice from control and 60 mg/kg/day treatment groups (n = 5 per group) were sacrificed and their tumors were digested to obtain single-cell suspensions for flow cytometry analysis.

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In separate experiments, 4T1 tumor-bearing BALB/c mice were administered 0

(vehicle control) or 60 mg sorafenib/kg/day (in 1:1 propylene glycol/water) for 6 days

imaging and in vivo PIT studies.

2.3. Flow cytometry analysis

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(days 0–5) by gavage. These mice were used for in vivo fluorescence and SPECT/CT

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Harvested tumors were minced into fragments, digested with 10 U/mL collagenase I, 400 U/mL collagenase IV, and 30 U/mL DNase (in phosphate buffered saline; PBS) for 1 h at 37°C, and then passed through a 70-µm cell strainer.

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Single-cell suspensions were stained with phycoerythrin (PE)-conjugated rat anti-mouse F4/80 and fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse

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CD206 antibodies (Sungene, Tianjin, China). Cells were sorted with an LSR-II flow cytometer (Becton Dickinson, Germany).

2.4. Synthesis and in vitro characterization of the anti-CD206 probe The CD206-targeting probe was generated using a previously described method [30]. Briefly, anti-CD206 antibody (clone C068C2, IgG2a; BioLegend, San Diego, 7

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CA) was mixed with IRDye700-N-hydroxysuccinimide (NHS) (LI-COR, Inc. Lincoln, NE) in sodium bicarbonate buffer (pH 8.3) at a 1: 7 mole ratio. After a 12 h

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incubation at 4°C, IRDye700-conjugated antibody (IRD-αCD206) was purified using a PD-10 desalting column (GE Healthcare, Piscataway, NJ) using PBS as the mobile

phage. The degree of labeling (dye/protein ratio) for IRD-αCD206 was calculated to

determined by sodium

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be approximately 4.1 based on UV measurements. The purity of IRD-αCD206 was dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE) and subsequent NIRF imaging with an IVIS optical imaging system (Xenogen, Alameda, CA). An isotype-matched control probe (IRD-IgG) was synthesized by conjugating rat IgG (IgG2a; BioLegend, San Diego, CA) with IRDye700-NHS using the same protocol.

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The immunoreactivity of IRD-αCD206 was evaluated relative to that of the parent antibody (αCD206) with a cell binding assay. Briefly, αCD206 was radiolabeled with Na125I to generate the radiotracer

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I-αCD206 using a previously described protocol

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[31]. The radiochemical purity of

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I-αCD206 after preparation was >98%.

CD206-positive [21] RAW264.7 cells were incubated with

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I-αCD206 in the

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presence of increasing concentrations of IRD-αCD206 and αCD206 in 96-well filter

plates. After incubating for 2 h at 4°C, the plates were washed using PBS, and the PVDF filters were collected and measured in a γ-counter. Data were fitted with

nonlinear regression using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) (n = 4).

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Singlet oxygen of IRD-αCD206 was measured using a singlet oxygen sensor green (SOSG) assay. Briefly, 1.0 µM SOSG (Invitrogen, Carlsbad, CA) was added to

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IRDye700 (1.0 µM) or IRD-αCD206 (1.0 µM IRDye700 equivalent concentration) solution, followed by irradiation with a 690-nm laser (Shanghai Laser & Optics Century Co., Ltd., Shanghai, China) for various times. SOSG fluorescence was

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measured with the IVIS optical imaging system (Xenogen, Alameda, CA) at an excitation wavelength of 460 nm and an emission wavelength of 520 nm. In the

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blocking experiments, the singlet oxygen quencher NaN3 (50 mM) was added to the solution and singlet oxygen molecules generated by IRD-αCD206 were detected as described above.

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2.5. In vivo NIRF imaging

Uptake of IRD-αCD206 by 4T1 tumors in the control or sorafenib-treated BALB/c mice was evaluated by NIRF imaging. Mice were treated with either the

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vehicle control or 60 mg sorafenib/kg/day for 6 days (days 0-5). On day 6, each mouse (n = 5 per group) was administered 0.5 nmol IRD-αCD206 by intravenous

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injection, and in vivo optical imaging was performed at 4, 8, and 24 h postinjection (p.i.). The uptake of IRD-αCD206 by tumor and muscle was determined by normalizing the fluorescence intensity of the tumor by the dose injected [32, 33]. After the last scan at 24 h p.i., mice were sacrificed. Tumors and major organs/tissues were dissected, weighed, and ex vivo NIRF imaged. Results are presented as the percent injected dose per gram (%ID/g). 9

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2.6. Small-animal SPECT/CT imaging

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4T1 tumor-bearing mice treated with either vehicle control or 60 mg sorafenib/kg/day for 6 days (n = 3 per group) administered 18.5 MBq of 125I-αCD206

by intravenous injection. After anesthetization with 2% isoflurane in oxygen, SPECT

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and helical CT scans of the mice were performed at 24 h p.i. on a NanoScan SPECT/CT imaging system (Mediso, Budapest, Hungary) [34, 35]. The tumor uptake

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of 125I-αCD206 was quantified using a previously described method [36, 37].

2.7. Evaluation of αCD206 microdistribution in sorafenib-treated tumors After six days (days 0-5) of sorafenib (60 mg/kg/day) treatment, 4T1

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tumor-bearing mice were injected intravenously on day 6 with 50 µg FITC-αCD206 (clone C068C2; IgG2a; Sungene, Tianjin, China). At 24 h p.i., tumors were harvested, cut on a freezing microtome, and immunolabeled with an antibody (Abcam,

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Cambridge, MA) against the M1/M2 macrophage marker F4/80 to determine

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macrophage localization.

2.8. In vivo PIT

4T1 tumor-bearing mice were treated with vehicle (control group, n = 12) or 60

mg sorafenib/kg/day for 6 days (days 0-5). On day 6, sorafenib-treated mice were randomized into four groups: (1) no treatment (sorafenib group, n = 12); (2) mice injected intravenously with 1 nmol IRD-αCD206 and then irradiated 8, 24, and 48 h 10

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p.i. at 70 J/cm2 with a 690-nm laser (PIT group, n = 12); (3) mice injected with 1 nmol IRD-αCD206 (probe only group, n = 12); (4) mice irradiated 8, 24, and 48 h p.i.

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at 70 J/cm2 with a 690-nm laser (light only group, n = 12). Tumor size was measured every other day. On day 10, five mice from each group were sacrificed and their tumors

were

harvested

for

histological

analysis

of

Ki67

and

terminal

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deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).

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2.9. Assessment of lung metastasis

At the end (day 16) of the in vivo PIT study, lung metastasis was assessed by high-resolution CT imaging and ex vivo examination. Mice were first scanned by CT to determine the position of the chest, and then the lungs were filled with 15% India

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ink via the upper trachea and fixed in Fekete’s solution (100 mL of 70% alcohol, 10 mL of 4% formalin, and 5 mL glacial acetic acid) for 48 h. Metastatic lesions on the black lung surface were counted. Lungs were then fixed with formalin, embedded in

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paraffin, cut into sections, and stained with hematoxylin and eosin (H&E).

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2.10. Immunofluorescence staining Ki67 and TUNEL fluorescence were determined in order to evaluate the effect of

PIT on tumor cell proliferation and apoptosis, respectively. For Ki67 staining, the frozen tumor sections were incubated with rabbit anti-Ki67 (Millipore, Billerica, MA) antibody for 1 h at room temperature and then visualized with dye-conjugated secondary antibodies under a Leica TCS-NT confocal microscope (Wetzler, 11

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Heidelberg, Germany). For TUNEL staining, the experiment was conducted by following the manual instruction of In Situ Cell Death Detection kit (Roche,

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Indianapolis, IN). After staining, 10 random views in the tumor slices were selected for the quantitative analyses. The tumor cell proliferation index and tumor apoptosis were calculated as the percentage of Ki67-positive nuclei and TUNEL-positive nuclei

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in relation to the total number of nuclei, respectively.

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2.11. Validation of PIT specificity

The specificity of IRD-αCD206 PIT for TAMs was evaluated in vitro and in vivo. For in vitro studies, CD206-positive RAW264.7 cells or CD206-negative [21] 4T1 tumor cells were incubated with 10 nM IRD-αCD206 for 1 h at 37°C. After washing

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with PBS, cells were irradiated at 0, 5, and 10 J/cm2 with a 690-nm laser. Propidium iodide (4 mg/mL; Sigma-Aldrich, St. Louis, MO) was added to stain dead cells, which were sorted by flow cytometry.

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For in vivo studies, 4T1 tumor-bearing mice were injected intravenously with PBS, 1 nmol of IRD-αCD206 or 1 nmol of IRD-IgG and then irradiated at 8, 24, and 48 h

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p.i. at 70 J/cm2 with a 690-nm laser (n = 3 per group). Mice were then sacrificed and

their tumors were dissected, cut with a freezing microtome, and immunolabeled with an antibody against F4/80 (to identify macrophages) and evaluated with the TUNEL assay (to identify apoptotic cells) as described above.

2.12. Statistical analysis 12

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Data were analyzed using the GraphPad Prism 5.0 software (GraphPad Software Inc.) and values are presented as mean ± SD. Statistical analysis was done using a

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1-way ANOVA for multiple groups and an unpaired Student’s t test. P values <0.05 were considered statistically significant.

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3. Results 3.1. Sorafenib treatment has no effect on 4T1 tumor growth

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We investigated the therapeutic efficacy of sorafenib in a 4T1 tumor model. After treatment for 6 days, sorafenib administered at doses of 20–60 mg/kg had negligible effects on tumors (Fig. 1A). There was no reduction in body weight at any dose, suggesting that the treatment was not toxic to mice (Fig. 1B).

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However, sorafenib increased the number of CD206+F4/80+ M2 macrophages and decreased the number of CD206−F4/80+ M1 macrophages relative to size-matched controls (Fig. 1C, 1D). These results indicate the repolarization of TAMs and their

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recruitment to sorafenib-treated tumors. In addition, the M2/M1 ratio was higher for sorafenib-treated as compared to control tumors (65.74 ± 9.17 vs. 45.17 ± 7.48, P

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<0.01; Fig. 1E).

3.2. Preparation of a TAM-targeted probe Due to increased M2 macrophage recruitment by sorafenib-treated 4T1 tumors, we hypothesized that CD206 could serve as a biomarker for 4T1 tumors after sorafenib treatment and that a CD206-specific probe would be useful for imaging and 13

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therapeutic targeting of sorafenib-treated tumors. We prepared the IRD-αCD206 probe by conjugating an anti-CD206 monoclonal antibody with a photosensitive

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near-infrared phthalocyanine dye (Fig. 2A). The purity of the probe was confirmed by SDS-PAGE and NIRF imaging (Fig. 2B). A competitive cell-binding assay with

CD206-positive RAW264.7 cells revealed that IRD-αCD206 (IC50 = 11.82 ± 3.44 nM)

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had CD206-binding affinity that was similar to that of the unmodified antibody (IC50 = 8.75 ± 1.97 nM; Fig. 2C).

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We measured the singlet oxygen molecules of IRD-αCD206 in PBS solution. As shown in Fig. 2D, E, the singlet oxygen generated after irradiation was increased for both IR700 and IRD-αCD206; these showed near-identical singlet oxygen generation. However, this was significantly inhibited by quenching with NaN3. These results

applications.

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confirmed the potential utility of IRD-αCD206 for in vitro and in vivo PIT

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3.3. In vivo targeting of tumors by IRD-αCD206 We investigated whether M2 macrophage recruitment by sorafenib-treated tumors

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can be noninvasively visualized by in vivo small-animal imaging using the IRD-αCD206 probe. Representative NIRF images of 4T1 tumor-bearing mice with or

without sorafenib treatment were examined at different time points after IRD-αCD206 injection (Fig. 3A). Subcutaneous tumors were detected from 4 to 24 h p.i. in both control and sorafenib-treated mice. However, the fluorescence intensity of IRD-αCD206 was significantly higher for sorafenib-treated as compared to control 14

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tumors at 4 (5.82 ± 0.38 % vs. 4.85 ± 0.15 %; P <0.01), 8 (9.20 ± 2.26 % vs. 5.98 ± 0.76 %; P <0.05) and 24 (9.17± 1.97 % vs. 5.73 ± 1.49 %; P <0.05) h p.i. (Fig. 3B).

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Tumor-to-muscle IRD-αCD206 ratios were also higher in sorafenib-treated tumors than in controls at 4 (3.13 ± 0.44 vs. 2.08 ± 0.15; P <0.01), 8 (4.98 ± 1.01 vs. 3.36 ± 0.32; P <0.01), and 24 (4.21± 0.49 vs. 2.60 ± 0.87; P <0.05) h p.i. (Fig. 3C). These

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results indicate that more IRD-αCD206 is taken up by sorafenib-treated as compared

to untreated tumors. The uptake of IRD-αCD206 in the sorafenib-treated tumors was

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also significantly higher than that of IRD-IgG at all-time points examined (P <0.05; Fig. S1), demonstrating the CD206-specific targeting of IRD-αCD206. To validate the in vivo NIRF imaging results, tumors were analyzed by ex vivo NIRF imaging and small-animal SPECT/CT. As shown in Fig. 3D, E, IRD-αCD206

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uptake by organs was similar between sorafenib-treated and control mice; however, probe uptake was greater in tumors treated with sorafenib (7.73 ± 1.07 %ID/g) as compared to controls (4.71 ± 0.29 %ID/g; P <0.05).

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Radionuclides are more quantitative than fluorescent dyes, and their in vivo distribution can be accurately and non-invasively quantified by SPECT imaging. We

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validated the results obtained by NIRF imaging of IRD-αCD206 by evaluating its

radioactive counterpart

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I-αCD206, which was predominantly taken up by the liver

(Fig. 4A) due to hepatic clearance. The probe showed increased accumulation in

tumors in sorafenib-treated mice compared to that in the control mice (Fig. 4A). Tomographic imaging revealed a variable distribution of

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I-αCD206 in

sorafenib-treated 4T1 tumors (Fig. 4B), reflecting the heterogeneous localization of 15

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TAMs. The quantification results confirmed that the uptake of

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I-αCD206 in the

sorafenib-treated tumors was significantly higher than that in the control tumors (P

To confirm the targeting of IRD-αCD206 or

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<0.01; Fig. 4C). I-αCD206 to TAMs in

sorafenib-treated tumors, we examined tumor tissue from sorafenib-treated 4T1

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tumor-bearing mice 24 h after injection of FITC-αCD206 for expression of F4/80

rather than CD206 in order to eliminate the possibility of competitive binding

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between the anti-CD206 antibody and FITC-αCD206. Note that the anti-CD206 antibody used in FITC-αCD206 was the same clone (C068C2) as that used in IRD-αCD206 or

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I-αCD206. As shown in Fig. 4D, most areas positive for

FITC-αCD206 showed F4/80 immunoreactivity, indicating that IRD-αCD206 and I-αCD206 were specifically localized in tumors. These results confirm that both

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probes can be used to image TAMs in vivo.

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3.4. IRD-αCD206 PIT prevents growth and metastasis of sorafenib-resistant tumors IRD-αCD206 PIT of the control tumors without sorafenib treatment showed a

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remarkable suppression on tumor growth (Fig. S2), which confirms the antitumor effect of PIT. However, the therapy efficacy IRD-αCD206 PIT is generally limited due to the low expression of CD206 in control tumors. We next investigated whether IRD-αCD206 could be used for in vivo PIT studies of sorafenib-treated tumors (Fig. 5A), which is supposed to express much higher CD206 compared to the control tumors (Fig. 1C, D, E). As shown in Fig. 5B, a time-dependent increase in tumor size 16

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was observed in 4T1 tumors treated with vehicle, sorafenib, sorafenib + light, and sorafenib + IRD-αCD206 only. In contrast, a single injection of IRD-αCD206

relative to the other groups from days 10 to 16.

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followed by irradiation at 8, 24, or 48 h p.i. significantly delayed tumor growth

We next carried out Ki67 immunolabeling and the TUNEL assay to assess tumor

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cell proliferation and apoptosis, respectively. As shown in Fig. 5C, the proliferation

index was decreased whereas the apoptosis index was increased in the sorafenib +

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IRD-αCD206 PIT as compared to control groups (P <0.001; Fig. 5D, E), demonstrating the efficacy of IRD-αCD206 PIT for treating sorafenib-resistant 4T1 tumors.

Subcutaneous injection of 4T1 tumor cells in mice results in metastasis to the

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lungs [21]. We therefore assessed whether IRD-αCD206 PIT can inhibit metastasis of sorafenib-resistant tumors by CT imaging. In control, sorafenib, sorafenib + light only, and sorafenib + IRD-αCD206 only groups, evident metastatic lesions were detected in

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the lungs; these were negligible in mice in the sorafenib + IRD-αCD206 PIT group (Fig. 6A). These findings were confirmed upon gross examination of lung lesions and

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by staining lung tissue sections with H&E (Fig. 6B, C). Mice in the sorafenib + IRD-αCD206 PIT group had fewer tumors than those in the control, sorafenib,

sorafenib + light only, and sorafenib + IRD-αCD206 only groups (P <0.05; Fig. 6D). These results suggest that TAM-specific PIT can inhibit tumor metastasis to the lungs.

3.5. IRD-αCD206 PIT targets TAMs in sorafenib-resistant tumors 17

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Although IRD-αCD206 PIT prevented lung metastasis of sorafenib-treated 4T1 tumors, the inhibitory effect on subcutaneous tumors was limited; that is, the

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treatment did not completely prevent tumor growth (Fig. 5B). We hypothesized that one reason for the low efficacy is that target cells (i.e., M2 macrophages) constitute a

very small population within the tumors, and photoactivation of IRD-αCD206 is

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targeted to CD206-positive areas. We therefore compared the cell-ablating capacity of

the probe in CD206-positive and -negative cells. Radiation dose-dependent toxicity

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was observed in CD206-positive RAW264.7 cells (Fig. 7A). However, there was no apoptosis observed in CD206-negative 4T1 cells after irradiation (Fig. 7A). Tumor-bearing mice were then subjected to IRD-αCD206 PIT, and apoptotic areas of tumor tissues were examined. As shown in Fig. 7B, C, a significantly greater

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number (P <0.01) of apoptotic cells were detected in IRD-αCD206 PIT as compared to control and IRD-IgG PIT-treated tumors. Importantly, most apoptotic (TUNEL-positive) nuclei in IRD-αCD206 PIT-treated tumors were associated with

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macrophages showing F4/80 expression at the cell surface. It should be noted that apoptotic tumor cells that are engulfed in macrophages may also be stained by

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TUNEL and then colocalized with F4/80. However, an almost undetectable cell (both tumor cells and macrophages) apoptosis was observed in the control tumor that was not treated with IRD-αCD206 PIT (Fig. 7B, C). Taken together, these data

demonstrate that IRD-αCD206 PIT can specifically destroy macrophages in the tumor.

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4. Discussion In this study we synthesized a probe (IRD-αCD206) targeting sorafenib-resistant

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tumors and investigated whether it can be used to visualize TAM recruitment and for PIT. We found that TAM recruitment to tumors treated with sorafenib can be detected

by IRD-αCD206 NIRF imaging, and that IRD-αCD206 PIT inhibited subcutaneous

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4T1 tumor growth and lung metastasis.

We used a 4T1 syngeneic tumor mouse model for in vivo sorafenib treatment. By

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measuring tumor growth after 6 days of sorafenib treatment, no statistical difference between the tumor sizes with or without sorafenib treatment was observed. In recent studies, Farsaci, et al. [38] observed that 7 days of sorafenib treatment had limited effect on the growth of 4T1 tumors, and only a long-term treatment could lead to the

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tumor responses to sorafenib. Hammond, et al. [39] also demonstrated that sorafenib treatment did not prolong the survival of 4T1 tumor-bearing mice compared to the vehicle control group. These results suggest that 4T1 tumors are resistant to sorafenib

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treatment.

It is thought that tumor hypoxia following sorafenib treatment plays an essential

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role in the development of resistance to the drug [7, 40, 41]. We found that sorafenib had no effect on tumor growth after 6 days of treatment, but increased hypoxia relative to control tumors (Fig. S3), which can lead to the recruitment and activation of various myeloid cells including TAMs [42]. Several studies have demonstrated the recruitment of macrophages after anti-angiogenic therapy or chemotherapy [2, 43]. Our study focuses on M2-type macrophage, which is one of the key factors for tumor 19

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progression and therapy resistance. The observed increase in M2 macrophage infiltration and CD206 expression in 4T1 tumors treated with sorafenib suggested that

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CD206 could serve as a marker for TAM infiltration of tumors as well as a target for PIT. In vivo NIRF imaging revealed the accumulation of TAMs in tumors after

sorafenib treatment. Given the limitations of planar optical imaging for deep tissue

analog of IRD-αCD206, namely

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imaging and quantification of fluorescence intensity, we synthesized a radiolabeled

I-αCD206, and evaluated its distribution by

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small-animal SPECT/CT imaging. The results were in accordance with the optical imaging data, and demonstrated the localization of

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I-αCD206 in 4T1 tumors. We

also confirmed that FITC-αCD206 specifically labeled TAMs. FITC-αCD206 showed only partial overlap with the F4/80 (marker of both M1 and M2 macrophages [44])

macrophages.

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signal, which is likely due to the fact that FITC-αCD206 is specific to M2 and not M1

Metastasis is one of the major causes of mortality in cancer patients; evidence

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suggests that TAMs promote distant tumor metastasis [11, 44]. By in vivo CT and ex vivo examination, we observed that the metastasis of subcutaneously inoculated 4T1

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tumor cells to the lungs was significantly blocked by depletion of TAMs by IRD-αCD206 PIT. These results demonstrated the anti-metastatic effect of

TAM-targeted PIT. Despite robust anti-metastatic effect, the efficacy of IRD-αCD206

PIT in subcutaneous 4T1 tumors treated with sorafenib was sub-optimal, as tumor growth was not entirely eliminated (Fig. 5B). There are two possible reasons for these results. Firstly, IRD-αCD206 targeted TAMs, which represent a small subset of cells 20

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in 4T1 tumors, resulting in low uptake of the probe by the tumor, which in turn reduces the effects of PIT; that is, irradiation eliminated only TAMs and not tumor

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cells. Secondly, like PDT, the effectiveness of PIT depends on the photosensitizer, light, and oxygen [45]; therefore, the hypoxic tumor microenvironment (Fig. S3) of sorafenib-treated 4T1 tumors may reduce the effectiveness of PIT due to a lack of

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oxygen.

After sorafenib treatment and subsequent CD206-targeted PIT, it did not cause an

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evident tumor sensitivity to the second rounds of sorafenib treatment (Fig. S4). These results suggest that IRD-αCD206 PIT has limited effect on the reversal of 4T1 tumor resistance to sorafenib. Chen et al. [7] demonstrated that one of the mechanisms underlying sorafenib resistance is the increased tumor hypoxia after sorafenib

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treatment. Increased hypoxia results in the recruitment and activation of multiple myeloid and lymphoid immune suppressor cells, including M2-type macrophages, myeloid-derived suppressor cells, and T-regulatory cells. Therefore, depletion of

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M2-type macrophages only (as we used in this study by IRD-αCD206 PIT) may not be sufficient to overcome tumor resistance to sorafenib.

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Radiotherapy or chemotherapy with drugs other than sorafenib can also lead to the

recruitment of TAMs by resistant tumors [2, 46, 47]. As such, the present findings can be extended to PIT of tumors treated with other therapies. In addition, several recent studies have shown the synergistic effect of sorafenib in combination with immune checkpoint inhibitors for enhanced antitumor efficacy by activating the function of cytotoxic T lymphocytes and boosting host antitumor immunity [7, 48]. PDT has also 21

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been reported to be effective on immunopotentiation [45]. Therefore, future studies investigating the combination of PIT and immune checkpoint inhibition to overcome

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tumor resistance to sorafenib therapy is highly desirable.

5. Conclusion

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The results of this study demonstrated that several rounds of sorafenib treatment can lead to M2 macrophage polarization in 4T1 tumors. The recruitment of TAMs by

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tumors was noninvasively visualized by NIRF imaging of the IRD-αCD206 probe. Upon irradiation, IRD-αCD206 PIT inhibited the growth of subcutaneous 4T1 tumors and prevented their metastasis to the lungs. These results suggest that TAM-targeted

Acknowledgments

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PIT is a promising strategy for treating tumors that are resistant to conventional drugs.

This work was supported, in part, by the “973” program (2013CB733802 and

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2011CB707705), the National Natural Science Foundation of China (81471712, 81222019, 81125011, 81371614, and 81420108019), the Beijing Natural Science (7132131

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Foundation

and

7132123),

and

the

Beijing

Nova

Program

(Z121107002512010).

Appendix A. Supplementary data Supplementary data related to this article can be found at http://xxxx.

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Figure Legends

Fig. 1. Sorafenib treatment has no effect on 4T1 tumor growth in vivo but leads to M2 macrophage polarization. (A, B) Tumor growth curves (A) and changes in body

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weight (B) of 4T1 tumor-bearing BALB/c mice treated daily with vehicle or indicated doses of sorafenib for 6 days. Inset, schedule of sorafenib treatment. (C)

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Immunofluorescence staining of murine CD206 in 4T1 tumor tissues after vehicle (control) or sorafenib (20, 40, and 60 mg/kg daily for 6 days) treatment. (D, E) Representative dot plots (D) and M2-to-M1 ratios (E) from flow cytometry analyses of F4/80 and CD206 expression by 4T1 tumor cells treated with vehicle or sorafenib sorafenib (60 mg/kg/day for 6 days). **, P <0.01.

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Fig. 2. Synthesis and in vitro characterization of IRD-αCD206. (A) Illustration of the synthesis of IRD-αCD206 for TAM targeting. (B) SDS-PAGE and NIRF imaging of

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IRD-αCD206 and αCD206. (C) Comparison of the CD206-binding affinities of IRD-αCD206 and αCD206 with the competition binding assay using 125I-αCD206 as a

radiotracer in CD206-positive RAW264.7 cells. (D, E) Singlet oxygen generation by

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IRDye700 and IRD-αCD206 with or without 50 mM NaN3 quenching after irradiation

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for indicated periods, as determined by the SOSG assay.

Fig. 3. Uptake of IRD-αCD206 by control or sorafenib (60 mg/kg/day for 6 days)-treated 4T1 tumors. (A) Representative NIRF images at 4, 8, and 24 h after IRD-αCD206 injection. Tumors are indicated with circles. (B, C) Quantitative

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analysis of uptake (B) and tumor-to-muscle accumulation ratios (C) of IRD-αCD206 in control or sorafenib-treated tumors. (D) Representative images of organs dissected from 4T1 tumor-bearing mice (with or without sorafenib treatment) sacrificed 24 h

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after intravenous injection of IRD-αCD206. (E) Quantitative analysis of IRD-αCD206

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distribution in dissected organs shown in panel D. *, P <0.05; **, P <0.01.

Fig.

4.

Macrophage-specific

localization

of

CD206-specific

probes

in

sorafenib-treated 4T1 tumors. (A) Representative small-animal SPECT/CT imaging of

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I-αCD206 24 h post-injection in control or sorafenib (60 mg/kg/day for 6

days)-treated 4T1 tumor-bearing BALB/c mice. Tumors are indicated with circles. (B) Representative 1-mm-thick transaxial slices though a 4T1 syngeneic tumor. (C) 30

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Comparison of the quantified uptake of

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I-αCD206 in the control and

sorafenib-treated tumors. (D) Distribution of FITC-αCD206 in sorafenib-treated 4T1

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tumor tissues harvested from mice 24 h p.i. Green, FITC-αCD206; red, rhodamine corresponding to F4/80; blue, DAPI-stained nuclei. **, P <0.01.

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Fig. 5. IRD-αCD206 PIT inhibits the growth of sorafenib (60 mg/kg/day for 6 days)-treated 4T1 tumors in BALB/c mice. (A) Schematic illustration of the PIT

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protocol in mice. (B) Growth curves of control or sorafenib-treated 4T1 tumors after treatment with light only, IRD-αCD206 (probe) only, or IRD-αCD206 PIT. (C) Histological

analysis

of

proliferating

and

apoptotic

cells

in

control

or

sorafenib-treated 4T1 tumor tissue after indicated treatments, as determined by Ki67

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immunolabeling and the TUNEL assay, respectively. (D, E) Quantitative analysis of Ki67- (D) and TUNEL- (E) positive cells shown in panel C. *, P <0.05; **, P <0.01;

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***, P <0.001.

Fig. 6. IRD-αCD206 PIT inhibits lung metastasis of sorafenib (60 mg/kg/day for 6

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days)-treated 4T1 tumors in BALB/c mice. (A–C) Representative images from CT scanning (A) and of India ink-filled lungs (B) and H&E-stained lung sections (C) from 4T1 tumor-bearing mice (on day 16) after indicated treatments. Lungs in the CT images and tumor metastases in the H&E-stained lung slices are indicated by dashed circles. Tumor metastases in the India ink-filled lungs are indicated by arrows. (D) Quantitative analysis of lesions in lungs shown in panel B. *, P <0.05. 31

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Fig. 7. Macrophage-specific cytotoxicity of IRD-αCD206 PIT in vitro and in vivo. (A)

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Flow cytometry analysis of dead CD206-positive RAW264.7 or CD206-negative 4T1 cells treated with PBS or IRD-αCD206 PIT (0, 5, and 10 J/cm2). (B) TUNEL and F4/80 immunolabeling for identification of apoptotic cells and macrophages,

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respectively, in 4T1 tumor tissues harvested from mice treated with PBS, IRD-IgG or

IRD-αCD206 (irradiated at 8, 24, and 48 h p.i. at 70 J/cm2 for all groups). (C)

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Quantitative analysis TUNEL-positive cells shown in panel B. **, P <0.01.

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